Porphyromonas gingivalis is the key etiological pathogen of periodontal disease, a chronic oral inflammatory condition that affects over 14% of the global population. P. gingivalis possesses several potent virulence factors—including gingipains, hemagglutinins, and fimbriae—that drive microbial dysbiosis. To restore homeostasis, nitric oxide (NO) is endogenously produced by immune cells to eliminate the infection, but disease persistence necessitates the use of therapeutic agents. The multiple antibacterial mechanisms of NO minimize the potential for antimicrobial resistance compared to traditional antibiotics, making exogenous NO delivery a potential attractive strategy for combating P. gingivalis. The objective of this study was to evaluate the effect of exogenous NO on the virulence of P. gingivalis, including NO's capacity to influence biofilm formation and dispersion, gingipain activity and production, invasion of host cells, and mitigation of immune responses. Nitric oxide effectively modulated the virulent behavior of anaerobic P. gingivalis via transnitrosylation and nitrosative stresses, reducing the pathogen's ability to form biofilms and gingipain activity. Within the anaerobic niche, NO exposure alters the pathogen's ability to export gingipains, thereby significantly reducing its potential to invade host cells and evade the immune system. This work highlights the therapeutic potential of exogenous NO as a promising therapeutic to alter periodontal disease through its multimodal effects on the virulence of P. gingivalis.

The topical treatment of acne vulgaris by a single therapeutic is difficult given the multifaceted causes of the disease. Treatment with photo-responsive nitric oxide (NO) donors and light represents an attractive alternative to conventional therapeutics due to the potential for multimodal therapeutic action against downstream acne processes. Herein, a topical hyaluronic acid (HA) formulation was developed to facilitate localized NO delivery from mesoporous silica nanoparticle (MSN) NO donors upon blue light exposure, while simultaneously providing anti-inflammatory activity. The NO release was tunable (0.09-0.51 µmol NO mg-1) depending on wavelength, with shorter wavelengths and greater irradiances providing more efficient photolytic liberation. Antibacterial activity and cytocompatibility were directly modulated by photolytic NO release and payload, where NO was determined to be the primary antibacterial agent and large, initial NO bursts led to preservation of cell viability. Incorporation of the MSN-based NO donors into an HA formulation afforded additional metabolic and immunomodulatory behavior in vitro, demonstrating potential to decrease sebum production and inflammation in acne vulgaris. Blue light-mediated NO release from the HA formulation presents a multifunctional therapeutic platform that addresses key contributing downstream factors of acne.

Objectives:This study investigated the antibacterial action of photo-liberated nitric oxide (NO) from primary and tertiary S-nitrosothiol-modified mesoporous silica nanoparticles (1° RSNO-MSNs and 3° RSNO-MSNs, respectively) against planktonic periodontal pathogens and ex vivo subgingival periodontal biofilms. Materials and methods:The antibacterial and antibiofilm efficacy of photo-liberated NO release from 1° RSNO-MSNs and 3° RSNO-MSNs was evaluated as a function of wavelength (405 and 455 nm), irradiance (400-1000 mW cm-2), and time (0-15 min) against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and ex vivo subgingival biofilms. Time course, biofilm dispersion, and biofilm eradication assays were employed to evaluate the synergistic impact of antibacterial blue light therapy (aBLT) and photo-liberated NO. Results:The addition of photo-initiated NO release with aBLT significantly improved antibiofilm activity, with irradiation of 405 nm at 1000 mW cm-2 demonstrating the most efficacious NO liberation from both 1° RSNO-MSNs and 3° RSNO-MSNs. While aBLT demonstrated antibacterial activity alone, the addition of NO was crucial for biofilm dispersal and eradication. Conclusions:The in vitro and ex vivo results demonstrate, for the first time, the utility of a dual-action aBLT and NO-releasing system for treating periodontal pathogens and biofilms.

The foreign body response (FBR) limits continuous glucose monitor longevity to 10 - 15 d because of chronic inflammation and collagen capsule formation surrounding the implant site. Nitric oxide (NO), a multifunctional bioactive molecule, has previously demonstrated potent FBR mitigation abilities. Here we evaluated the impact of polyurethane sensor membranes doped with a range of NO-release profiles achieved by primary and tertiary S-nitrosothiol-functionalized mesoporous silica nanoparticles (1° and 3°RSNO MSNs) using an in vitro approach. Experiments were performed using NO concentrations correlating to those known to be released at 1, 2, 4, and 6 weeks for each NO-releasing RSNO-MSN system. The NO-release kinetics and durations achieved by 1°/3°RSNO-MSN-doped sensor membranes proved effective at regulating pro- and anti-inflammatory cytokine secretion, inhibiting foreign body giant cell formation, preserving fibroblast migration, and reducing collagen production when evaluated using NO concentrations known to be present at 6 weeks. Such results suggested 1°/3°RSNO-MSN-doped sensor membranes have the potential to extend glucose sensor lifetime beyond the previously achieved 4 weeks. Indeed, both 1°/3°RSNO- and 3°RSNO-MSN-coated glucose sensors maintained stable mean absolute relative difference values over 6 weeks, with 1°/3°RSNO-MSN coated sensors trending lower, demonstrating the promise of extended NO-release for maintained sensor function over time.

Acne vulgaris (acne) is a common skin disorder associated with significant psychosocial impact. Current clinical therapies include topical and systemic antibiotics, benzoyl peroxide, and retinoids. While moderately effective, these clinical therapies fail to target all four major pathogenic causes of acne and are associated with painful side effects. Nitric oxide (NO), an endogenous signaling molecule, represents a promising alternative to conventional acne treatments due to its innate antibacterial and immunomodulatory functions. As NO is highly reactive, macromolecular NO donors are required for its controlled, solution-phase delivery. Prior work has utilized silica nanoparticle scaffolds to store and deliver NO, with the silica scaffold being considered inert. Herein, NO-releasing hyaluronic acid (HA), an endogenously produced biopolymer, was modified with NO donors to enable a dual-action therapeutic capable of addressing the pathogenic factors responsible for acne development. The molecular weight of these HA derivatives proved important with respect to bactericidal activity against Cutibacterium acnes and ability to modulate keratinocyte proliferation, sebum production, and inflammation.

Targeted therapeutic delivery for treating bacterial infections remains underutilized in most pharmaceutical interventions. Existing therapeutics (i.e., antibiotics) are often systematically administered despite the presence of localized infection, leading to both off-target toxicity and suboptimal bacterial clearance with limited efficacy against biofilms. The overuse of antibiotics has resulted in increased antimicrobial resistance, creating a need for alternative interventions that are unlikely to confer resistance. Nitric oxide (NO), an endogenous mediator produced by macrophages and other immune cells in response to infection, elicits broad spectrum antibacterial and antibiofilm activity. The use of exogenous NO donors, alone or as conjugated ligands to macromolecular scaffolds, has proven effective in treating anatomical targets, including dermal wounds, dental infections, and pulmonary conditions, in a localized manner. In this perspective, we provide an overview of the recent advancements in NO-releasing biomaterials, highlighting design strategy and antimicrobial action across diverse anatomical sites.

The exogenous delivery of nitric oxide (NO) as a therapeutic is challenging due to NO's high reactivity and short half-life. Owing to NO's many physiological functions, the development of NO-releasing materials for sustained and long-term NO delivery has been a focus of much research. Herein, we report the synthesis of an organosilane bestowing a tertiary thiol (3 degrees SH) moiety derived from (3-aminopropyl)trimethoxysilane and N-acetyl-d-penicillamine thiolactone (NAP-thiolactone). The resulting tertiary thiol-bearing organosilanes (NAPTMS) were covalently tethered to mesoporous silica nanoparticles (MSNs) to generate 3 degrees SH-enriched MSNs (MSN-NAP) that, when nitrosated, formed NO-releasing mesoporous silica nanoparticles (MSN-SNAP). The morphological and thermomechanical properties of MSN-NAP and MSN-SNAP proved suitable as potential therapeutic materials, with NO payloads (1.39 +/- 0.15 mu mol mg-1) approximately ten times greater than previously reported tertiary S-nitrosothiol (3 degrees RSNO) systems. In contrast to other reports, MSN-SNAP exhibits a sustained NO-release profile for >24 h under physiological conditions. The MSN-SNAP thus represents a promising NO-releasing prodrug for various biomedical applications that require sustained NO delivery at low concentrations (pM to nM).

Taking advantage of their innate roles as antibacterial strategies, the dual activity of photobiomodulation (PBM) and nitric oxide (NO) was combined to provide a tunable, on-demand chronic wound therapeutic. S-nitrosothiol-modified mesoporous silica nanoparticles (RSNO-MSNs) were doped into polyurethane (PU) to demonstrate preliminary utility as an antibacterial wound dressing treatment for chronic wounds. Photoinitiated and resultant NO-release kinetics and payloads were evaluated at 405, 430, and 530 nm for multiple irradiances. The use of photons and the NO-releasing MSNs against common chronic wound pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, proved to be highly bactericidal. Cytocompatibility of the treatment was confirmed using human epidermal keratinocytes, a representative skin cell line.

Traditional wound dressings are applied to cover and protect a wound from further injury and infection to provide time for healing. Thus, a wound dressing with bioactivity that has pro-wound healing and antibacterial action represents a promising alternative for traditional wound dressings. In this study, a blend of poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) was electrospun into fiber mats, coated with hyaluronic acid (HA), and functionalized with N-diazeniumdiolate nitric oxide (NO) donors to develop a dressing capable of clearing bacterial infection and promoting wound healing in vivo. These electrospun fibers were highly porous with uniform diameters and capable of releasing large quantities of NO (∼0.6 μmol mg-1) for up to 12 h. The mats displayed swelling capacities of over 300% and resisted degradation up to 10 d, making them useful for long-term wound healing. The NO-releasing electrospun fibers demonstrated potent antibacterial activity against Pseudomonas aeruginosa in vitro and improved bacterial clearance, reduced inflammation, and promoted wound healing in an in vivo punch-biopsy wound model. Together, these data demonstrate that electrospun fibers represent a promising option for use as bioactive wound dressings.

The burn wound healing process is dysfunctional. Conversion of previously undamaged tissue to damaged can prolong the healing process with increases in morbidity and mortality of patients. Therapies that utilize anti-inflammatory molecules, like nitric oxide (NO), have been proposed to modulate this inflammatory process. In low doses, NO has been shown to downregulate innate immune cells, stimulate angiogenesis, and promote fibroblast activity at the wound site. NO is physiologically unstable on its own. However, when coupled with other matrices, it can be released in a controlled state. Our collaborators utilize an amine (N-(2-hydroxyethyl)ethylenediamine; HEDA)-modified Chondroitin-sulfate C (CSC) scaffold that delivers NO to tissue, with minimal toxicity, and possess non-burn wound regenerative properties. We hypothesize that in combination with pluronic F127 organogel to aid efficient and stable delivery to wound tissue, CSC-HEDA/NO will modulate wound conversion and reduce healing time for burn wounds in mice. Wildtype female C57BI/6 mice weighing 18-22g underwent a 20% total body surface area full-thickness cutaneous contact burn or sham injury (n=6) with appropriate anesthesia. Analgesia was provided through the length of the experiment. Mice were treated immediately after injury with 100ul of CSC-HEDA/NO directly applied to the wound surface, and every three days thereafter for 14 days. We evaluated rate of wound closure, grade of inflammation and wound score, and wound conversion. At 14 days, mice were euthanized and wounds were removed for RNA isolation. nanoString immune transcriptomic analyses (Mouse Immunology CodeSet) and corresponding Ingenuity Pathway Analysis (IPA) were performed. We found no significantly altered conversion between the groups, and observed decreased closure in the CSC-HEDA/NO group at 8, 11, 12, 13 and 14 days post-injury compared to the untreated group. Other wound scores were unchanged between groups. However, transcriptomic analysis showed a significant (P< 0.05) upregulated of 14 immune genes compared to untreated wounds, including IL6RA (+2.3 fold-change (Fc)), NFKB2 (+1.8fc) and ITGAX (+2.1fc). IPA demonstrated significant alteration in canonical immune pathway activation with significant reprogramming (induction) of the IL-4/IL-13, Th1/Th2 activation, and FAK signaling pathways. Studies of the role of NO in wound healing by multiple groups has demonstrated diverse pro- or anti-inflammatory effects, and these data reveal a significant induction of inflammatory reprogramming in burn wound tissue. Histology data are still pending which will further reveal mechanism of the decreased wound closure. We are currently investigating the use of CSC-HEDA/NO during concurrent bacterial infection. NO has been suggested in studies to improve burn wound outcomes with often paradoxical outcomes. These data add to the body of knowledge. N/A

While nitric oxide (NO) release from polyurethane (PU) sensor membranes has shown promise as a foreign body response (FBR) mitigation strategy to enhance the performance of implantable glucose sensors, its utility is ultimately limited by release duration. Further improvement is envisioned by combining electrospun fibers with NO release. Electrospinning process parameters that produce average fiber diameters of 670 and 1460 nm as the outer membrane of NO-releasing glucose sensors, are developed to not impact NO-release or sensor performance. An in vivo evaluation in a diabetic porcine model demonstrates a reduced inflammatory response for 670 versus 1460 nm fibers. This benefit appears to continue with a robust pro-wound healing response beyond the NO-release duration. At short periods (i.e., 11-d post-implantation), FBR mitigation is attributed to NO release and not the presence of fibers. Still, no negative effects are observed with the 670 nm fibers in this acute phase of the FBR. Taken together, the tissue response data demonstrate 670 nm fibers as a promising long-term FBR mitigation strategy.

Nontuberculosis mycobacteria (NTM) are ubiquitous, opportunistic pathogens that cause severe respiratory infection, primarily in elderly and immunocompromised populations. The second most prevalent NTM pathogen, Mycobacterium abscessus, is considered the most refractory due to its fast growth rate, intracellular survivability, and antibiotic resistance. Treatments are thus sparse and generally ineffective, promoting antibiotic resistance upon chronic use. Nitric oxide (NO) is an endogenously produced free radical that exerts antimicrobial effects against pathogens via several mechanisms of action, and as such, it is unlikely to elicit resistance. Methyl tris diazeniumdiolate (MD3) is a small-molecule NO-releasing prodrug that is capable of sustained NO release, making it an attractive candidate as an antimicrobial therapeutic; however, its triple negative charge makes cellular uptake unlikely. As liposomes enable cellular uptake, their use as an MD3 delivery system may further enhance the utility of NO release for treating intracellular NTM infections. Herein, liposomal formulations were evaluated as a function of pH and buffer composition and optimized for MD3 loading to enable the delivery of bactericidal levels of NO. Planktonic studies with two clinically relevant morphotypes of M. abscessus revealed that lower pKa liposomal systems employ a better antimicrobial efficacy. Prevention and eradication assays revealed that liposomal MD3 significantly improves biofilm inhibition compared to nonliposomal MD3 and was capable of eradicating biofilm bacteria at 4 mg mL-1. Liposomal MD3 and MD3 had similar reductions in intracellular bacterial load, achieving at least a three-log reduction at relevant concentrations. Fluorescence spectroscopy over 24 h demonstrated that liposomal encapsulation increased the intracellular concentration of a membrane-impermeable fluorophore by 3.4-fold. Confocal microscopy was used to visualize the increase in the number of cells containing intracellular NO and the sustained presence of NO within the cell, confirming that liposomal MD3 increases small-molecule internalization.

Immune dysregulation, which occurs when the balance of the immune system is compromised, is responsible for many inflammatory and chronic diseases, including those associated with non-resolving infection. Nitric oxide (NO), an endogenous signaling molecule, represents an alternative therapeutic to standard of care interventions as it has the potential to address both chronic infection and inflammation. Hyaluronic acid (HA) is a naturally occurring biopolymer involved in native immune responses, yet its properties as a NO-delivery vehicle can be multifactorial based on its molecular weight. Herein, a library of NO-releasing HA derivatives varying in molecular weight, alkylamine modification, conformation, and NO-release kinetics was synthesized to assess the influence of these properties on biocompatibility and the immunomodulatory activity of macrophages. The NO-release kinetics were dependent on the HA molecular weight, alkylamine modification, and conformation; however, these properties did not influence biocompatibility. All HA derivatives, regardless of their properties, were able to bind to their cognate receptors, but differences in HA properties affected the strength of signaling and macrophage polarization.

Non-tuberculosis mycobacteria (NTM) can cause severe respiratory infection in patients with underlying pulmonary conditions, and these infections are extremely difficult to treat. In this report, we evaluate a nitric oxide (NO)-releasing prodrug [methyl tris diazeniumdiolate (MD3)] against a panel of NTM clinical isolates and as a treatment for acute and chronic NTM infections in vivo. Its efficacy in inhibiting growth or killing mycobacteria was explored in vitro alongside evaluation of the impact to primary human airway epithelial tissue. Airway epithelial tissues remained viable after exposure at concentrations of MD3 needed to kill mycobacteria, with no inherent toxic effect from drug scaffold after NO liberation. Resistance studies conducted via serial passage with representative Mycobacterium abscessus isolates demonstrated no resistance to MD3. When administered directly into the lung via intra-tracheal administration in mice, MD3 demonstrated significant reduction in M. abscessus bacterial load in both acute and chronic models of M. abscessus lung infection. In summary, MD3 is a promising treatment for complex NTM pulmonary infection, specifically those caused by M. abscessus, and warrants further exploration as a therapeutic.

Melanoma is an aggressive skin cancer notorious for high levels of drug resistance. Additionally, current treatments such as immunotherapies are often associated with numerous adverse side effects. The use of nitric oxide (NO) may represent an attractive treatment for melanoma due to NO’s various anticancer properties, unlikeliness to foster resistance, and limited toxicity toward healthy tissues. The anticancer effects of chemical NO donors have been explored previously but with limited understanding of the needed characteristics for exerting optimal antimelanoma activity. Herein, the in vitro therapeutic efficacy of three macromolecular NO donor systems (i.e., cyclodextrin, mesoporous silica nanoparticles, and hyaluronic acid) with tunable NO-release kinetics was explored by evaluating skin permeation along with toxicity against melanoma and healthy skin cells. Cytotoxicity against melanoma cells was dependent on NO payload and not donor identity or NO-release kinetics. In contrast, cytotoxicity against healthy cells was primarily influenced by the macromolecular NO donor, with cyclodextrin- and hyaluronic acid-based NO donors having the highest therapeutic indices. In vitro skin permeation was influenced by both the size and charge of the NO donor, with smaller, more neutral donors resulting in greater permeation. A Pluronic F127 organogel was optimized for the delivery of a cyclodextrin-based NO donor. Delivery of the NO donor in this manner resulted in increased in vitro skin permeation and reduced tumor growth in an in vivo model.

Chronic wounds impact 2.5% of the United States population and will continue to be a major clinical challenge due to increases in population age, chronic disease diagnoses, and antibiotic-resistant infection. Nitric oxide (NO) is an endogenous signaling molecule that represents an attractive, simple therapeutic for chronic wound treatment due to its innate antibacterial and immunomodulatory function. Unfortunately, modulating inflammation for extended periods by low levels of NO is not possible with NO gas. Herein, we report the utility of a NO-releasing glycosaminoglycan biopolymer (GAG) for promoting wound healing. GAGs are naturally occurring biopolymers that are immunomodulatory and known to be involved in the native wound healing process. Thus, the combination of NO and GAG biopolymers represents an attractive wound therapeutic due to these known independent roles. The influence and contribution of chondroitin sulfate C (CSC) modified to facilitate controlled and targeted delivery of NO (CSC-HEDA/NO) was evaluated using in vitro cell proliferation and migration assays and an in vivo wound model.

Drug resistance and off-target toxicity are two of the greatest challenges to chemotherapeutic melanoma treatments. Nitric oxide (NO) represents an attractive alternative to conventional therapeutics due to its numerous anticancer properties and low probability of engendering resistance. As NO is highly reactive, macromolecular NO donors are needed for the controlled and targeted delivery of NO for therapeutic applications. Herein, mesoporous silica nanoparticles (MSNs) coated with hyaluronic acid (HA) were developed as a NO delivery system to facilitate controlled delivery to cancer cells through both passive and active targeting via the enhanced permeation and retention effect and directed binding of HA with CD44 receptors, respectively. The aminosilane modification, HA concentration, and HA molecular weight were systematically evaluated to facilitate the MSN coating and NO loading. The hydrodynamic diameter and dispersity of the nanoparticles increased after HA coating due to the hydrophilic nature of HA, with greater increases observed at higher HA molecular weight. Lower starting concentrations of HA and aminosilanes with longer alkyl chains favored more efficient HA coating. Faster NO-release kinetics and lower NO payloads were observed for the HA-coated MSNs relative to uncoated MSNs. However, the localized delivery of NO to cancer cells through the active targeting conferred by HA increased levels of oxidative stress and induced mitochondria-mediated apoptosis in melanoma cells. Cytotoxicity was also evaluated against human dermal fibroblasts, with the use of 6 kDa HA-coated MSNs resulting in the greatest therapeutic indices. Enhanced internalization of HA-coated nanoparticles into melanoma cells versus uncoated nanoparticles was visualized with confocal microscopy and quantified by fluorescence spectroscopy. In total, HA-coated MSNs represent a promising NO delivery system for potential use as a chemotherapeutic for skin melanomas.

Improving the utility of biomedical devices implanted in subcutaneous tissue by modulating the innate immune response common to these implants is of great interest to improve their utility. Uncontrolled, most biomedical devices produce an immune reaction known broadly as the foreign body response (FBR), which ultimately isolates the device from the native tissue. The use of electrospun fibers to create a porous surface that promotes tissue in-growth and regeneration represents a new paradigm in FBR modulation. A vast number of parameters can be adjusted in the electrospinning process to tune the type and quality of the resulting electrospun matrix, which in turn has varying outcomes with respect to the FBR. In this review, the fabrication and utility of electrospun fiber scaffolds for mitigating the FBR are described, with details of how fiber properties and surface modifications alter immune response for specific biomedical applications.

Klebsiella pneumoniae is considered to be a critical public health threat due to its ability to cause fatal, multi-drug-resistant infections in the bloodstream and key organs. The polysaccharide-based capsule layer that shields K. pneumoniae from clearance via innate immunity is a prominent virulence factor. K. pneumoniae also forms biofilms on biotic and abiotic surfaces. These biofilms significantly reduce penetration by, and antibacterial activity from, traditional antibiotics. Nitric oxide (NO), an endogenous molecule involved in the innate immune system, is equally effective at eradicating bacteria but without engendering resistance. This study investigated the effects of NO-releasing small molecules capable of diverse release kinetics on the capsule and biofilm formation characteristics of multiple K. pneumoniae strains. The use of NO donors with moderate and extended NO-release properties (i.e., half-life > 1.8 h) inhibited bacterial growth. Additionally, treatment with NO decreased capsule mucoviscosity in K. pneumoniae strains that normally exhibit hypermucoviscosity. The NO donors were also effective against K. pneumoniae biofilms at the same minimum biocidal concentrations that eliminated planktonic bacteria, while meropenem showed little antibacterial action in the same experiments. These results represent the first account of exogenous NO affecting biomarkers involved in K. pneumoniae infections, and may therefore inform future development of NO-based therapeutics for treating such infections. (c) 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Pathogenic fungi are an increasing health threat due to the rise in drug resistance. The limited number of antifungals currently available and growing incidence of multi-drug-resistant fungi has caused rising healthcare costs and a decreased quality of life for patients with fungal infections. Nitric oxide (NO) has previously been shown to act as an antimicrobial agent, albeit with a limited understanding of the effects of the NO-release kinetics against pathogenic fungi. Herein, the antifungal effects of four nitric oxide-releasing small molecules were studied against the pathogenic fungi Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus, to demonstrate the broad-spectrum antifungal activity of NO. A bolus dose of NO was found to eradicate fungi after 24 h, where nitric oxide donors with shorter half-lives achieved antifungal activity at lower concentrations and thus had wider selectivity indexes. Each NO donor was found to cause a severe surface destruction of fungi, and all NO donors exhibited compatibility with currently prescribed antifungals against several different fungi species.